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A Afzal, LC Shaw, S Caballero, EA Ellis, MB Grant; The Development of Hammerhead Ribozymes that Specifically Cleave the A2B receptor mRNA . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3711.
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Purpose:Vascular eye diseases such as retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR) are characterized by the abnormal growth of blood vessels on the retina. Tissue hypoxia and ischemia initiate a series of events that lead to angiogenesis. Potential mediators of angiogenesis include nucleosides such as adenosine. Adenosine modulates a variety of cellular functions by interacting with specific cell surface receptors (A1, A2A, A2B, A3). To date, the lack of a potent, selective A2B adenosine receptor inhibitor has hampered the characterization of the cellular functions modulated by the activation of this receptor. Our goal was to produce a hammerhead ribozyme that would specifically cleave the A2B receptor mRNA and selectively reduce the expression of this protein in human retinal endothelial cells (HRECs) and in in vivo angiogenesis models. Methods:We have performed multiple turnover kinetic analysis on two hammerhead ribozymes (A2BRz1 and A2BRz2) designed to specifically cleave the mouse A2B receptor mRNA. The more catalytically active ribozyme has been cloned into a recombinant Adeno Associated Viral vector (rAAV) for subsequent packaging into AAV and for cell transfection and in vivo experimental analysis. We have used this AAV construct to transfect HRECs and used the modified Boyden chamber assay to examine ribozyme effects. Results:In vitro kinetic analysis demonstrated that the A2BRz2 ribozyme was the more efficient ribozyme with a Vmax (542 nM min-1) and a kcat (36.1 min-1) approximately 20-fold higher than the values obtained for A2BRz1 ribozyme. These values are greater than values we have obtained with other ribozymes (Shaw, et al., 2001), and suggest that both of these ribozymes would be effective ribozymes in vivo. This ribozyme has been cloned into a rAAV vector and we have also cloned an inactive version of the A2BRz2 that will be used to separate the catalytic activity from the antisense activity of the active ribozyme. We are now in the process of testing these AAV-ribozyme constructs HRECs and in a mouse model for ROP. Preliminary data from the transfection of HRECs indicate that A2BRz2 selectively effects the expression of the A2B receptor. Preliminary data in the ROP mouse model shows a reduction in retinal angiogenesis after the intravitreal injection of the ribozyme construct. Conclusion:Our data suggest that the A2BRz2 will effectively reduce the A2B receptor mRNA in vivo and provide a tool for the selective inhibition of this receptor.
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